In some mutants we have looked at we see a defect in Ape1 processing under non-starvation conditions but a very mild detect under nitrogen starvation conditions. We interpreted that as our gene of interest being involved in the cvt pathway. But when we looked at Atg8-GFP we saw a complete block under nitrogen starvation conditions. This suggests a block in non-selective autophagy. But why, then, is Ape1 only partially defective under nitrogen starvation? We have seen this in papers but it is never well-explained.
That it sort of puzzling.
To speculate: perhaps your mutant has a large (but not quite 100%) defect in core autophagy - i.e. both selective and non-selective. By Atg8-GFP this might look like a complete block. But perhaps the Ape1 processing assay is a little bit more sensitive (I don't know this to be the case, but it would seem plausible to me, since Ape1 is presumably more stable in the vacuole than GFP), and therefore it shows up as only a partial block.
Ape1 transport to the vacuole is greatly increased during starvation conditions when non-selective autophagy is induced; this could be why the Ape1 processing looks like a full block in growing conditions but only a partial block in starvation.
Again, the idea is that your mutant is allowing just a tiny bit of autophagy (selective and non-selective, equally) - not enough to see by Ape1 processing in growing conditions, nor enough to see by Atg8-GFP processing under any condition, but enough to see a little bit of Ape1 processing under starvation conditions, when there is more Ape1 transport.
Does that sound plausible? I suppose it depends on exactly what your blots look like.
Ape1 is also a vacuolar hydrolase so you should take this into account, but are also different ways to induce autophagy. Nitrogen starvation is a specific way to induce autophagy and it depends on 2 recently characterized proteins Npr1 and Npr2. These signal upstream of TOR and do not seem to be involved in other autophagy induced pathways. There was a paper on this in MBoC not too long ago. You should be able to find it without much trouble.
Have you tried it with nitrogen deprivation and rapamycin treatment side by side? Is there any difference?
Taras,
Yes, that's the same thing was thinking - except you explained it better.
And yes, of course, GFP-ATG8, not ATG8-GFP. Good catch.
Thanks to all who answered and shed light on this. Very helpful. And Taras, well explained and makes sense. Sorry for the typo - it is of course GFP-Atg8p.
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